Aquaporin 8ab is required in zebrafish embryonic intestine development

The aquaporin 8 (AQP8) is a small integral membrane protein that selectively transports water and other small uncharged solutes across cell plasma membranes. It has been demonstrated that AQP8 is ubiquitously present in various tissues and organs of mammals, and participates in many physiological and pathological processes. Recent studies showed that AQP8 is highly expressed in the columnar epithelial cells of mammalian colonic mucosa facing lumen, indicating that AQP8 plays potential roles in the physiology and pathophysiology of gastrointestinal tract. However, the role of AQP8 during gastrointestinal tract development is unclear. In the present study, RT-PCR results reveal that the zebrafish genome encodes three kinds of aqp8s ( aqp8aa, aqp8ab, and aqp8b). We use whole mount in situ hybridization to describe aqp8 genes spatiotemporal expression pattern, and the results show that aqp8ab mRNA is detectable mainly in the zebrafish embryonic intestine. To reveal the details of aqp8ab distribution, histological sections are employed. Transverse sections indicate that aqp8ab mRNA expression is more intense in the layer lining the intestinal cavity. Knockout of aqp8ab using the CRISPR/Cas9 system induces intestine development defects and abnormal formation of intestinal lumen. In addition, aqp8ab mRNA significantly rescues the intestine defects in the aqp8ab mutant. These results indicate that aqp8ab is required in the intestine development of zebrafish.


Introduction
The primary functions of the gastrointestinal (GI) tract are digestion, absorption, excretion, and protection [1]. To perform these functions, the GI epithelium in close contact with the GI lumen forms a physical and biochemical barrier to separate the GI lumen from the underlying tissues [2][3][4]. Water, electrolytes and nutrients move across the barrier in a regulated manner either between epithelial cells or across the apical membrane of epithelial cells. Proper function of the GI tract is essential for supporting life. A growing body of evidence suggests that defects in intestinal barrier function are associated with several different human diseases, including inflammatory bowel disease (IBD), diarrhea, celiac disease, and irritable bowel syndrome (IBS) [5]. However, the mechanisms responsible for GI physiology and function and the critical steps in GI disease pathogenesis are still unclear.
Transcellular water flux primarily occurs via aquaporins (AQPs) in the mammalian digestive system [6]. The AQPs, which belong to major intrinsic protein (MIP) family, are integral membrane proteins that selectively transport water and other small uncharged solutes across cell plasma membranes [7][8][9]. Until now, at least eight AQPs have been found to be expressed in various tissues in the mammalian GI tract, and they are distributed in a distinct regional and subcellular manner [10][11][12]. The special locations of AQPs are relevant to their functions. Numerous studies have demonstrated that the alterations of AQPs expression and distribution in the GI are associated with gut disorders and diseases [13][14][15].
It has been demonstrated that AQP8 is highly expressed in the columnar epithelial cells of mammalian colonic mucosa facing lu-men, indicating that AQP8 plays potential roles in the physiology and pathophysiology of gastrointestinal tract [16][17][18][19]. In contrast to mammals, three kinds of aqp8s (aqp8aa, aqp8ab, and aqp8b) are have been reported in zebrafish [20,21]. The aqp8s have been found to be expressed in the digestive system of zebrafish and possibly play critical roles in controlling or maintaining water homeostasis. However, the role of zebrafish aqp8s in GI organ morphogenesis throughout the embryonic development remains unclear.
Previous studies have shown that intestinal anatomy and architecture in zebrafish is closely related to mammals, and it has emerged as a valuable model organism for genetic research of vertebrate organ morphogenesis [22][23][24][25][26]. The zebrafish intestinal tract is divided into three main segments: the intestinal bulb, the midintestine and the posterior intestine. Instead of a stomach, the intestinal bulb acts as the major site of lipid and protein digestion [27].
In the present study, we used the zebrafish model to analyze the role of aqp8s in intestine development. RT-PCR and whole mount in situ hybridization results revealed that aqp8aa was highly expressed in the vascular system, aqp8ab was observed in the intestine, and aqp8b was detected in the kidney. To reveal the details of aqp8ab distribution, histological sections were employed. Transverse sections indicated that aqp8ab mRNA expression was more intense in the layer lining the intestinal cavity. Knockout of aqp8ab using CRISPR/Cas9 system induced intestine development defects and abnormal formation of intestinal lumen. In addition, aqp8ab mRNA significantly rescued the intestine defects in the aqp8ab mutant. These results indicate that aqp8ab is required in the intestine development of zebrafish. Furthermore, our results further promote the understanding of the cellular and molecular mechanisms responsible for GI organ morphogenesis and patterning.

Zebrafish tissues and embryos
The zebrafish (AB line) used in this study was obtained from China Zebrafish Resource Centre (Wuhan, China) and maintained in Jiangsu key laboratory of Neuroregeneration of Nantong University (Nantong, China). Zebrafish embryos were obtained through natural mating and maintained at 28.5°C. Embryonic stages were defined as described previously [28]. At 24 h post-fertilization (hpf), embryos were treated with 0.2 mM 1-phenyl-2-thio-urea (PTU), a tyrosinase inhibitor commonly used to block pigmentation and aid visualization of zebrafish development. Embryos for whole mount in situ hybridization (WISH) were collected at various stages, fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4°C or 2 h at room temperature, washed with PBST (PBS plus 0.1% Tween-20), dehydrated in methanol, and stored at -20°C until use. Embryo development stages less than 24 hpf were dechorionated after fixation, prior to storage. For zebrafish over 4 days post-fertilization (dpf), immobilization by submersion in ice water (5 parts ice/1 part water, 0-4°C) for at least 20 min to ensure death by hypoxia. For embryos ≤ 3 dpf, development was terminated using bleach. Addition of bleach solution (sodium hypochlorite 6.15%) to the culture system water at 1 part bleach to 5 parts water. The embryos remained in this solution at least five minutes prior to disposal to ensure death. This procedure was approved by the Administration Committee of Experimental Animals of Nantong University (Approval ID: 20180608-Z001). The pain perception has not developed at these earlier stages, so this is not considered a painful procedure. All animal experiments were car-ried out in accordance with the NIH Guidelines for the Care and Use of Laboratory animals (http://oacu.od.nih.gov/regs/index.htm). This study covers the 3Rs (refinement, replacement, and reduction).

RNA extraction, reverse transcription, and PCR
Embryos and tissues were homogenized and frozen using Trizol reagent (Invitrogen, Carlsbad, USA) and stored at -80°C. Total RNA was extracted according to the manufacturer's instruction. RNA (1 μg) was reverse-transcribed into cDNA using Transcriptor First Strand cDNA Synthesis kit (Roche, Basel, Switzerland) according to the manufacturer's instruction. Synthesized cDNA was stored at -20°C. All PCR amplifications were carried out in a total volume of 50 μL using 2×Taq PCR SuperMix (Trans, Beijing, China) according to the manufacturer's instruction.

aqp8ab mRNA preparation and injection
The cDNA containing the coding sequence of the aqp8ab gene was cloned into PCS2+ vector and then was transcribed in vitro using the mMESSAGE mMACHINE SP6 kit (Ambion) after the recombinant plasmid linearized with NotI (NEB, Beverly, USA), and then the capped mRNA was purified using the RNeasy Mini kit (Qiagen). aqp8ab gene mRNA (2 nL) was injected at 50 ng/μL into the 1-cell stage embryos.

Histology
For sectioned histological analysis, the whole-mount in situ hybridized embryos in 100% glycerin were replaced by Tissue-Tek OCT (Sakura, Tokyo, Japan) two times for 30 min each to remove the glycerin. Subsequently, embryos were embedded in plastic molds containing OCT and the embryos orientation was adjusted with a needle. The embryos were then sectioned with a CM1900 UV (Leica, Wetzlar, Germany) at 14 μm. After being dried for 4 to 5 h at 37°C, the sectioned samples were washed three times (10 min each) with PBS and stored with a mounting medium.

Microscopy analysis
The results of whole-mount in situ hybridization and pictures in bright field were documented with an Olympus DP60 camera on an Olympus stereomicroscope MVX10 (Olympus, Tokyo, Japan), Leica imaging system on a Leica compound microscope and Zeiss SteREO Discovery V20 microscope with a Zeiss AxioCam HRc camera (Zeiss, Oberkochen, Germany). For confocal imaging of intestine development in zebrafish embryos, they were anesthetized with egg water (0.16 mg/mL tricaine/1% 1-phenyl-2-thiourea; Sigma, St Louis, USA) and embedded in 0.6%-0.8% low melting agarose. Confocal imaging was performed with a Nikon TI2-E-A1 HD25 laser scanning confocal microscope (Nikon, Tokyo, Japan).

Statistical analysis
Image analysis was performed using Imaris microscopy image analysis software (Bitplane AG, Zurich, Switzerland). Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, USA). Student's t-test was used for comparison between groups, and P<0.05 was considered statistically significant.

aqp8ab is expressed in developing intestine of zebrafish
To determine the role of aqp8ab in the GI development, we investigated the expression of aqp8ab during embryonic development in zebrafish via WISH using antisense RNA probe. We successfully cloned three kinds of aqp8s (aqp8aa, aqp8ab, and aqp8b) using RT-PCR from zebrafish embryos. To determine the tissue-specific expression pattern of aqp8s, we performed WISH analysis using digoxigenin (DIG)-labeled antisense probe. The results showed that aqp8aa was highly expressed in vascular system, aqp8ab was observed in the intestine, and aqp8b was detected in the kidney (Supplementary Figure S1).
To investigate the expression dynamics of aqp8ab during embryonic development, we performed detailed WISH analysis using aqp8ab probe. In the 14, 24, 36 or 48 hpf stage embryo, the hybridization signal of aqp8ab was not detected. In the 72 hpf stage embryo, weak expression of aqp8ab was observed in zebrafish intestine ( Figure 1A). The expression of aqp8ab in intestine was increasingly maintained during zebrafish early embryo development. No hybridization signal of aqp8ab was found in embryos via WISH using sense RNA probe (Supplementary Figure S2).
The aqp8ab expression patterns were confirmed using the probe of intestinal marker intestine fatty acid binding protein (ifabp; fabp2-ZFIN) from Peng's lab [30,31], and the result was consistent with the aqp8ab probe expression patterns. In the 4, 5 and 6 dpf stage embryos, clear hybridization signal of ifabp was seen in zebrafish intestine ( Figure 1B).

aqp8ab mRNA expression is more intense in the layer lining the intestine cavity
To reveal the details of aqp8ab distribution in the zebrafish intestine, we conducted histological section experiments on embryos. Transverse sections showed that in the anterior and middle region of the intestinal tract at 4 dpf, the aqp8ab mRNA expression was presumably confined to the epithelial cells lining the intestinal cavity (Figure 2A,B). By 5 dpf, the aqp8ab mRNA expression was more intense in the layer lining the intestinal cavity ( Figure 2D-F).

Establishment of aqp8ab knockout mutant line by CRISPR/Cas9 system
In order to examine whether aqp8ab is required for the development of intestine, the CRISPR/Cas9 system was utilized to generate a series of aqp8ab mutants in zebrafish. aqp8ab sgRNA mRNA and Cas9 mRNA were co-injected into the 1-cell stage embryos to knock out the genes. We chose the target sites in the third exon of zebrafish aqp8ab and identified the effective sgRNA ( Figure 3A). To examine the mutation type, we extracted genomic DNA from embryos at 24 hpf with injected mRNAs and performed PCR to amplify the target region. PCR products were sequenced and sequence analysis revealed 11 types of mutations ( Figure 3B). The remaining sibling of these G0 embryos was raised to adulthood. The G0 founders carrying somatic mutations were out-crossed with wildtype fish to obtain F1 offspring. We identified 3 types of mutations among the adult F1 fish by PCR amplification and sequencing with tail fin-clipped genomic DNAs ( Figure 3C). The mutated alleles included a 1-bp deletion, a 4-bp deletion and another 4-bp deletion, which all resulted in reading frame shift and premature translation termination ( Figure 3D). According to the previous protein sequences analysis, the truncated proteins lose three essential domains including NPA, H7 and H8 [20]. Moreover, the abnormal mRNAs will probably be degraded by the mechanism of NMD, which results in no protein production.

aqp8ab deletion induces abnormal phenotype in embryonic development
To examine the effect of the mutation during intestine development, we observed embryo development at different stages in bright field. The deletion of aqp8ab had no significant effect on embryonic development in the early stage (data not shown). At stage 3 dpf, the embryo sizes of the aqp8ab mutants were decreased and had slight pericardial edema phenotype ( Figure 4A). At 4 and 5 dpf, the de-
To detect the effects of aqp8ab deletion on the intestinal morphology, we carried out WISH using ifabp probe. The mutants had a normal intestinal tract at 3 dpf ( Figure 4B). At 4 and 5 dpf, compared with the control group, the deletion of aqp8ab induced zebrafish embryo intestinal bifida, and the intestines were deformed ( Figure  4B). These results suggested that aqp8ab might play a role in zebrafish embryonic intestine development.

Loss of aqp8ab impairs zebrafish intestinal lumen formation
To further detect the function of aqp8ab in intestine development, the embryos after WISH were sectioned ( Figure 5 and Supplementary Figure S3). In the control group, transverse sections through the trunk at four different regions demonstrated a single intestinal tract, in which a lumen was already formed (Figure 5B,C). Cross-sections revealed double independent intestinal tracts with no lumen or a small lumen at 4 and 5 dpf, compared with the control group ( Figure  5B,C and Supplementary Figure S3). These results suggested that embryonic intestinal lumen development might be impaired.

aqp8ab mRNA injection can rescue the abnormal phenotype induced by aqp8ab gene deficiency
To further determine whether the abnormal phenotype observed in the aqp8ab mutants is due to the loss of function of aqp8ab rather than to nonspecific effects, we performed the rescue experiment. The mixture of sgRNA (500 ng), Cas9 mRNA (1500 ng) and aqp8ab mRNA (250 ng) were injected into the 1-cell stage embryos. It was found that the embryos co-injected with aqp8ab mRNA can rescue the intestine development defects (Figure 6). These results showed that intestine defects are specifically caused by inactivation of aqp8ab.

Discussion
AQPs are a group of membrane transport proteins that assemble in cell plasma membranes as tetramers and facilitate the passage of water and other small solutes, such as glycerol, urea, and carbon

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AQP8 in zebrafish embryonic intestine development dioxide. To date, 13 mammalian AQPs have been identified, which are present in various tissues and organs with functions including from acid secretion [32], renal collection ducts and body water homeostasis [33], blood-brain barrier permeability [34], immune system regulation [35] and tumor biology involvement [36]. AQPs are physiologically essential in mammals.
Previous studies showed that AQP8 is expressed in mammalian digestive system, such as salivary glands, pancreas, liver, small intestine, and large intestine [10,37]. The distributions of AQP8 are relevant to its function, indicating that AQP8 might play potential physiological roles in the digestive system. A recent study revealed that AQP8 facilitates the transport of water and small molecules on the apical membrane of rat small intestine [38]. However, the role of AQP8 during gastrointestinal tract development is still unclear. Here, we cloned three kinds of aqp8s (aqp8aa, aqp8ab, and aqp8b) in zebrafish, and our results showed that aqp8ab is essential for intestinal organogenesis. The expression profile of aqp8ab was detected, revealing that it is present in various stages during zebrafish embryogenesis and is mainly expressed in the embryonic intestine. The deletion of aqp8ab induced intestine development defects and abnormal formation of intestinal lumen.
In a previous study, AQP8 was found to localize to the apical membrane of the colonic intestinal epithelial cells (IECs), and down-regulation of epithelial AQP8 may impair water resorption in active collagenous colitis (CC), resulting in watery diarrhea [19]. The function of AQP8 in the water absorption and secretion of small intestine and colon is limited in AQP8-knockout mice model [17].
In the murine colonitis model, the expression level of AQP8 is reversely correlated with the occurrence of inflammation and injury [38]. These data show that the alterations of AQP8 expression may impair the passage of transcellular water and induce inflammatory bowel disease, suggesting that AQP8 is a potential drug target for the treatment of intestinal disorders and diseases.
Intestinal epithelial cells are polarized and form a luminal surface by their apical membranes. In a previous study, the interaction of cortical membrane organizer EMR-1 and AQP8 was found to propel lumen extension by transluminal flux, suggesting a direct morphogenesis effect of water-channel-regulated fluid pressure [39]. The deletion of aqp8ab in intestinal epithelial cells impairs the transport of transcellular water and results in the imbalance of water homeostasis, thereby inducing an intestine defect and abnormal formation of intestinal lumen.
In summary, aqp8ab affects intestine development in embryos of zebrafish, and the intestinal lumen formation is affected.

Supplementary Data
Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

Funding
This work was supported by the grants from the National Nature Science Foundation of China (No. 81870359), the Science Foundation from Jiangsu Province (Nos. BK20180048, 17KJA180008, BRA2019278 and SWYY-048) and the Science Foundation from Suqian (Nos. WSW-302 and S201723).

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AQP8 in zebrafish embryonic intestine development

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AQP8 in zebrafish embryonic intestine development